Biochimica et Biophysica Acta 1854 (2015) 110–117

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Functional and dysfunctional conformers of human neuroserpincharacterized by optical spectroscopies and Molecular Dynamics

Rosina Noto a,1, Maria Grazia Santangelo a,1, Matteo Levantino b, Antonio Cupane b, Maria Rosalia Mangione a,Daniele Parisi a,c,2, Stefano Ricagno c, Martino Bolognesi c, Mauro Manno a,⁎, Vincenzo Martorana a

a Institute of Biophysics, National Research Council of Italy, Palermo, Italyb Department of Physics and Chemistry, University of Palermo, Palermo, Italyc Department of Biosciences, Institute of Biophysics CNR, Italy and CIMAINA, University of Milano, Milan, Italy

⁎ Corresponding author at: Institute of Biophysics, NatioUgo La Malfa 153, 90146 Palermo, Italy. Tel.: +39 091 68

E-mail address: mauro.manno@cnr.it (M. Manno).1 These authors equally contributed to the work.2 Present address: Institute of Integrative Biology, Unive

http://dx.doi.org/10.1016/j.bbapap.2014.10.0021570-9639/© 2014 The Authors. Published by Elsevier B.V

a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 June 2014Received in revised form 4 September 2014Accepted 3 October 2014Available online 6 November 2014

Keywords:NeuroserpinSerpinConformational diseaseFluorescenceMolecular DynamicsCircular dichroism

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, includingthe Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerizationof NSmutants. Herewe present an in vitro and in silico characterization of native neuroserpin and its dysfunction-al conformation isoforms: theproteolytically cleaved conformer, the inactive latent conformer, and the polymericspecies. Based on circular dichroism and fluorescence spectroscopy,we present an experimental validation of thelatent model and highlight the main structural features of the different conformers. In particular, emission spec-tra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopictool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latentserpins,we propose a structuralmodel for latent NS, for which an experimental crystallographic structure is lack-ing. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatialdistribution of intramolecular salt-bridges and hydrogen bonds.

© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/3.0/).

1. Introduction

Neuroserpin (NS) is an axonally secreted protein [1], belonging tothe Serpin family (SERine Protease INhibitor) [2]. NS is an inhibitor oftissue-type plasminogen activator, with a role in physiological pro-cesses [3] such as synaptic plasticity, memory, or sterol metabolism[4], as well as in pathological contexts, such as Alzheimer disease[5]. Site mutations in NS amino acid sequence cause an autosomaldominant dementia, known as Familial Encephalopathy withNeuroserpin Inclusion Bodies (FENIB) [6], related to aberrant deposi-tion of NS polymers [7–10]. Such pathology, characterized by an ev-ident genotype–phenotype correlation [11], is a striking example of aclass of conformational diseases, the serpinopathies, related to spe-cific serpin site mutations, as in the case of the most common α1-antitrypsin deficiency [12].

Neuroserpin shares the typical serpin fold characterized by a largefive stranded β-sheet (sA), partially covered by an α-helix (hF), and

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rsity of Liverpool Liverpool, UK.

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an exposed reactive central loop (RCL) that acts as a pseudo-substratefor the target protease [13]. Protease inhibition occurs when the RCL iscleaved at a specific site by the protease and is inserted as a new strandof the A β-sheet, while the protease remains covalently trapped as anacyl enzyme intermediate [14]. In the case of NS such a final complexis poorly stable: the protease is eventually released, while NS remainsin a stable loop-inserted “cleaved” isoform [15]. Alternatively, as forother serpins, NS may insert the uncleaved RCL into the A β-sheet,thus achieving the “latent” isoform, a permanently inactive state thatis more stable than the native one [16]. On the other hand, the so called“mouse-trap”mechanism, which is at the heart of the serpin inhibitionmechanism, can lead to linear protein polymer production, through theserial intermolecular exchange of RCLs among neighboring serpin mol-ecules [17]. The first polymerization mechanism proposed to explainpolymerization considered the insertion of the RCL of one moleculeinto the activated A β-sheet of a nearby molecule [18,19]. Recently,other models have been proposed, involving extensive domain swap-ping and major unfolding of the polymer forming molecules [20,21].

In recent studies, we showed how the structural details of dys-functional NS conformers, polymer and latent NS, depend upon ther-modynamic and environmental conditions [22,23], in keeping withother studies [24]. Further, we proposed that the mechanism of NS

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111R. Noto et al. / Biochimica et Biophysica Acta 1854 (2015) 110–117

polymerization is rate limited by the formation of an intermediate con-formation prone to dimerization [25,26], as in other serpins [27], and iscontrolled by a peculiar link between depolymerization and concomi-tant latentization [25]. Further, Onda and coworkers have recently iden-tified a refolding intermediate leading to the formation of polymersalike those formed by native NS [28].

Detailed knowledge on serpin structure and dynamic behavior(intermediate conformations, conversion to latency, polymerization)is fundamental to designmolecules for the treatment of diseases depen-dent on serpin polymerization [29]. However, the structure of the path-ological polymers is still a matter of debate [17], and the structuralinformation concerning a dysfunctional state such as the latent state isstill elusive, only a few crystal structures being available [30–34].

Beyond its relevance in human pathology, NS is an excellent modelfor biophysical studies of serpin polymerization due to its close structur-al homology with the archetypal serpin α1-antitrypsin (AAT) [15], butalso in view of its capability of forming polymers and latent conformersunder thermal stress [8–10,22–26] or under acidic conditions [24], evenin the wild-type form. Indeed, NS is relatively less stable than AAT, asmarked by the achievement of fragile NS oligomers [25], and by the la-bility of the tPA–NS complex [15]. The determination of the crystalstructures of the native NS [15,35] and of its cleaved form [15], alongwith recent results obtained by NMR and computer simulation [36],have helped in recognizing some of the key dynamical and structuralfeatures of NS.

Here, we extend the previous studies to all NS functional and dys-functional conformations, by performing in vitro and in silico character-izations of native, cleaved, latent and polymeric NSs.

In particular, far-UV circular dichroism and intrinsic fluorescencemeasurements highlight the existence of distinct conformational fin-gerprints for the different NS conformers, and at same time a close anal-ogy between the latent and cleaved NSs, in keeping with our model forthe latent NS. A key feature is given by the emission spectra of ionizedtyrosine residues, which are less contributed in the latent and cleavedform.

Moreover, we investigate the structural and dynamical differencescharacterizing the native, cleaved and latent conformers of NS, throughmolecular modeling techniques. In the absence of experimental 3Dstructures, a reasonable model is proposed for the latent NS, based onthe corresponding conformer of AAT [30] and on the cleaved NS [15].Our long equilibrium Molecular Dynamics (MD) simulations point outa hindered mobility and an enhanced stability (approximately 10–20 kcal mol−1 free-energy) of the latent and cleaved NS conformationsrelative to the native one, in part revealed by a salt-bridged network.

2. Materials and methods

2.1. Production of different neuroserpin conformers

RecombinantNSwas expressed and purified according to a previous-ly published protocol [15,28]. Protein concentration was measured byUVabsorptionat280nm(extinctioncoefficient:0.803cm−1mg−1ml−1,molecular mass: 46,280 g mol−1). Latent and polymer NSs were pro-duced by incubating a 20 μM solution of native NS at 55 °C for 2 h; thepolymers were subsequently separated by size exclusion chromatogra-phy (SEC; Appendix A, Supporting Fig. A1). Cleaved NS was obtainedby incubating a 200 μMsolution of native NS at 37 °C for 1 hwith trypsin(Sigma), applying a 1:10 protease–NS concentration ratio. The proteo-lytic reaction was blocked by prompt addition of soybean trypsin inhib-itor (Sigma) at the final concentration of 150 μM. CleavedNSwas furtherpurified by SEC. The clear-cut identification of NS conformers and thelack of native protein in the latent sampleswere assessed by native poly-acrylamide gel electrophoresis, which showed that the non-native con-formations migrate faster than native NS due to their more compactshapes [37] (Appendix A, Supporting Fig. A2).

2.2. Circular dichroism

Circular dichroism (CD) spectrawere recorded, using 0.01 cm quartzcuvettes, on a J-815 spectropolarimeter (Jasco, Tokyo, Japan) equippedwith a Peltier-type temperature-control system. The spectra were ac-quired with the average of 9 scans (3 nm bandwidth, 8 s response,10 nmmin−1 scan rate) and baseline-corrected by subtracting a bufferspectrum. The mean residue differential extinction coefficient Δεres, inM−1 cm−1 units, was calculated from the observed ellipticity θ, indegrees, by the expression Δεres = θ(Nresdc32.982)−1 [38], where d isthe path length in cm, Nres = 410 is the number of residues in therecombinant NS used, and c is the protein molar concentration: 20,21, 12.4 and 20 μM, for native, cleaved, latent and polymer NSs,respectively.

2.3. Intrinsic fluorescence

Fluorescence spectra were acquired at different excitation wave-lengths (λex) over a 260–520 nm emission wavelength (λ) range usinga 3 mm quartz cuvette and a Jasco FP-6500 spectrofluorimeter at roomtemperature (response 2 s, 1 nm excitation bandwidth, 3 nm emissionbandwidth, 100 nm min−1 scan rate), and baseline-corrected bysubtracting a buffer spectrum. All the spectra were corrected for the in-strument response (evaluatedwith an independentmeasurement of theemission spectrum of tryptophan in water [39,40]), and subtracted ofthe Rayleigh elastic peak. Emission spectra have been normalized withrespect to their integral and then scaled in order to fit the tail (above450 nm) of the emission spectrum measured with excitation at295 nm. The rationale of this treatment is that, upon excitation at295 nm, only tryptophan residues are excited. At the same time, tyrosineemission above 450 nm is negligible. The resulting Ī(λex;λ) spectra aredirectly related to the spectra STrp(λ) and STyr(λ) of Trp and Tyr residues,respectively: Ī(λex;λ) = STrp(λ) + STyr(λ)ATyr(λex), where ATyr(λex) isthe integral of Tyr spectra.

2.4. Molecular Dynamics simulations

The NS models corresponding to each state were solvated in rect-angular simulation boxes of 9.3 × 7.3 × 7.2 nm3 for native NS,6.8 × 7.9 × 8.6 nm3 for cleaved NS, and 6.7 × 7.7 × 8.7 nm3 for latentNS. Each system contained about 12,000–13,000 TIP3 water molecules[41],with 15potassium ions to neutralize the protein net charge, addingup to a total of 42,000–45,000 atoms. The water box was created to se-cure a solvent layer of 1 nm in each direction. The tautomeric form ofthe His residues appropriate for neutral pH was chosen with the helpof a VMD tool. After adequate minimization and equilibration steps(Appendix A, Supporting Table A1), MD trajectories were generated,using the NAMD2 package [42] and the Charm22 force field [41] inthe NPT ensemble at 300°K and atmospheric pressure; periodic bound-ary conditions were employed, Van der Waals and Coulomb interac-tions were truncated using a switch function at a cutoff value of 1 nm,while long-range electrostatic interactions were evaluated by theParticle Mesh Ewald method. The SHAKE algorithm was used to con-strain the bond lengths of heavy atoms, allowing the use of a 2 fs timestep. A simulation of the same system in the NVE ensemble was per-formed to check for energy conservation, to ensure that the equationsof motion were accurately solved.

2.5. Computational analytical tool

Different software packages and computational tools were used forthe analysis of secondary structure elements, Solvent Accessible SurfaceArea, hydrogen bonds, and essential modes (Appendix A, Table A2).

200 220 240Wavelength (nm)

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Fig. 2. CD spectra of NS conformers. (a) Far UV CD spectra of native (gray circles), cleaved(red circles), latent (green circles) and polymeric (blue circles) NSs; continuous blackcurves are the best fitting to the data. (b) Secondary structure contents reported as num-ber of residues (#) or as overall residue percentage (%), resulting from CD spectra fitting,for native (gray bars), cleaved (red bars), latent (green bars) and polymer (blue bars) NSs.

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3. Results and discussion

The serpin tertiary structure typically hosts three beta-sheets, labeledA, B and C, and nine alpha-helices. Fig. 1 shows a cartoon representationof NS in its native conformation, where such main secondary structurefeatures are highlighted. Three regions are known to be important forthe serpin conformational changes and are located at the top, center-top, and at the center of the A β-sheet respectively [13]. Such key regionsare conventionally identified as: (i) the hinge, considered a switch pointfor RCL insertion; (ii) the breach, the onset point for RCL insertion; and(iii) the shutter, behind the center of the A β-sheet containing also partof the s6B strand and the top of helix B.Wemonitored a fourth importantregion: (iv) the gate region, located between the turn linking the G helixwith the s3B strand (gate-turn1) and the turn linking strands s3C and s4C(gate-turn2). Insertion of the RCL into theAβ-sheetwithout any cleavage(inactivation or latentization) requires it to pass through the gate region.

3.1. Characterization of NS secondary structure and validation of the latentmodel

The secondary structure contents of each of the three monomericconformations (native, cleaved and latent), along with those of poly-meric NS, were addressed by far-UV CDmeasurements. The CD spectraof both cleaved and latent NSs show a less intense negative band around220 nmand209nm, relative to the spectrumof nativeNS, suggesting anincrease in β-sheet structure [38]. Conversely, the CD spectrum of poly-meric NS exhibits a band at lower wavelengths, typical of more disor-dered structures.

A more quantitative estimate was obtained by fitting the CD spectrausing the CDPro software package [43] (Fig. 2a), confirming a higher ex-tent ofβ-structure in the latent and the cleaved forms (Fig. 2b). Interest-ingly, the difference between the polymer and native NS structure is inkeeping with results previously reported by us and others [25,27,35],and consistent with the generally accepted idea that serpin polymersshould not involve extensively unfolded molecules [17,29].

3.2. Characterization of NS overall structure and identification of the latentconformation

We structurally characterized the different NS conformations bymeasuring the intrinsic fluorescence emission, which in proteins isdominated by tryptophan (Trp) and tyrosine (Tyr) residues [39].

Fig. 1.Three views of thehumanNS. The cartoon representation presents thenomenclature of sefront. Center: 90° rotation across the horizontal axis; the RCL is on the front. Right: 180° rotatiohighlighted in colors: A β-sheet (orange), B β-sheet (lime), C β-sheet (prune), RCL (green–blu

Wild-type NS hosts three Trp and fourteen Tyr residues. In order toseparate their spectral contributions, the emission spectra were mea-sured at two different excitation wavelengths: 295 nm, where only

condary structure elements. Left: the classical presentation of serpinswith Fα-helix on then around the vertical axis, with theα-helix F on the back. The main structural features aree), and F α-helix (cyan) and Ω-loop (red). Some key regions are circled.

image of Fig.�2

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Trp contributes, and 275 nm, close to the absorption maximum of bothTrp and Tyr.

The intrinsic fluorescence emission spectra of the different NSconformers, Ī(λex;λ), are displayed in Fig. 3a and b for the excitation at295 and 275 nm, respectively. Emission spectra were also measured atdifferent excitation wavelengths around the absorption peak (270 and280 nm), recording analogous behaviors, since the relative absorptionof Tyr and Trp is alike around the maximum (data not shown). Byobserving the second derivative minima (at the bottom of Fig. 3a,b)we note that the emission spectra are highly structured. Wemay singleout five main contributions, centered at 304, 318, 331, 344 and 360 nm,respectively (other minor bands could be considered at 310, 326 and352 nm). Note that the band at 360 nm is included to take into accountthe non-symmetric shape of the emission band and to correct the intrin-sic bias introduced by an analysis in terms of Gaussian components.These components match the scheme of discrete classes of Trp residuespredicted to be most probable in proteins [39,44], which include:(i) Class A, λm = 308 nm, buried Trp; (ii) Class S, λm = 316 nm, buriedTrp forming exciplexes (H-bonded complexes in the excited state);(iii) Class I, λm = 331 nm, buried Trp forming multiple exciplexes;(iv) Class II, λm = 341 nm, partially exposed Trp; and (v) Class III,λm = 351 nm, extensively exposed Trp. The analysis in terms ofGaussian components (Appendix A, Supporting Fig. A3) allows to quan-tify the contribution from each emission bands (Fig. 3c and d for the ex-citation at 275 and 295 nm, respectively).

The emission spectrum of polymeric NS exhibits a clear red-shiftrelative to the native NS, along with a conspicuous intensity quenching(although the latter effect is not evidenced in the normalized spectrashown in Fig. 3a,b). This indicates that the Trp residues of polymericNS are more exposed to the solvent than those in the monomeric

Fig. 3. Fluorescence spectra of NS conformers. Panels (a) and (b): emission spectra of native (b(a) and 275 nm (b). At the bottom of panels (a) and (b): second derivative of the emission splegend).

conformers, in agreement with previous studies by us and others[25,28]. At the opposite, the emission spectrum of cleaved NS is slightlyblue-shifted, due to the more pronounced band at 304 nm. This isevident from inspection of the 295 nm excitation Ī(λex = 295 nm; λ),and may be ascribed to a more constrained Trp environment, likelydue to a damping of conformational fluctuations, as evidenced by MDsimulations. The band at 304 nm may be due to Tyr emission, as evi-denced by the spectra with 275 nm excitation Ī(λex = 275 nm; λ)(Fig. 3b,d). This characteristic shoulder at 304 nm caused by both Trpand Tyr emission stands as a characteristic feature of cleaved NS,which allows distinguishing the cleaved conformers from the nativeone.

The latent emission spectrum with excitation at 295 nm is largelynot distinguishable from the native one. In particular, the low wave-length bands of native and latent NSs are comparable, suggesting neg-ligible perturbation of the Trp environments in terms of exposure orreducedmobility. Indeed, no evident spectral differences have been ob-served so far in the emission of different NS or serpin conformers, withthe exception of the trivial red-shift in polymers or partially foldedintermediates [35,45–47]. The picture changes at the 275 nm excitation.Here, the latent emission spectrum is closely similar to that of thecleaved one (Fig. 3b,d). Therefore, Tyr emission allows identifying thelatent conformation and marks a spectroscopic fingerprint for thisconformer. Latent and polymer NSs were also formed by incubation athigher temperature exhibiting the same spectroscopic features ofthose formed at 55 °C.

The ratios in the Gaussian band area at 304 and 331 nm for the na-tive, cleaved and latent NSs are 0.36, 0.51, and 44, respectively, at295 nm, while they increase to 0.66, 0.97, and 0.96 at 275 nm. In otherwords, in the latent and cleaved NSs the two bands become equivalent.

lack), cleaved (red), latent (green) and polymeric (blue) NSs, with excitations at 295 nmectra. Panels (c) and (d): fractions of the different Gaussian components (colors as in the

300 320 340 360 380emission wavelength (nm)

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114 R. Noto et al. / Biochimica et Biophysica Acta 1854 (2015) 110–117

An analysis-free parameter, unbiased by both instrumental calibrationand data normalization is the ratio R of the emission intensities at304 nm (with excitations at 275 and 295 nm) relative to the corre-spondent intensities at 331 nm: namely R = I (275 nm; 304 nm) /I(275 nm; 331 nm) ∙ [I(295 nm; 304 nm) / I(295 nm; 331 nm)]−1(see Table 1).

The R-values for the native, cleaved and latent NSs are R = 1.33,1.42, and 1.55 respectively. This parameter, along with the overallshape of the emission spectrum excited at 275 nm,may represent a use-ful tool for serpin studies, since it allows a possible non-invasive identi-fication of serpin monomeric conformers, besides the classical nativepolyacrylamide gel electrophoresis approach [37].

Fig. 4. Tyr emission spectra. Solid curves are native (black), cleaved (red), latent (green)and polymer (blue) NSs. Dotted line is a standard emission spectrum of Tyr inwater. Spec-tra are normalized to the maximum.

3.3. Tyrosine emission spectra and interaction network

The shape of the emission spectra of the NS conformers poses thequestion of the origin of the differences in Tyr emission. Since Tyr emis-sion is relatively insensitive to the local environment [39], it could beexcluded that the observed differences arise from a different solventexposure of Tyr residues. Also, although a Tyr–Trp resonant energytransfer should display a high efficiency in NS, this is not the origin ofthe spectral differences, since the Tyr spectra integrals have comparablevalues [48] (Appendix A, Supporting Fig. A4). Fig. 4 shows the Tyrspectra obtained by subtracting the spectra of Fig. 3a from the spectraof Fig. 3b: ĪTyr(λ) = Ī(λex = 275 nm; λ) − Ī(λex = 295 nm; λ). Thespectral shapes are wider than the typical Tyr spectrum in aqueous sol-vent, and such a broadening is enhanced in native and polymeric formsand reduced in cleaved and latent conformations. One may representthese spectra with a proper Tyr band centered at 304 nm and a secondband around 340 nm, which is typical of the emission of tyrosinate,which may depend on a decrease of the residue pKa in the excitedstate [39]. The proximity of a positive charge to the Tyr hydroxylgroup may also help lowering the intrinsic Tyr pKa promoting the for-mation of tyrosinate. The only basic residue having an average distancefrom a Tyr within 3 Å is Arg362 on the RCL, which is close to Tyr218 onthe s3C strand in native NS, and displaced upon inactivation in thecleaved and latent NSs. Other differences in the location of Tyr residuesbetween native and cleaved NSs may be found in residues 291 (onstrand 2C) and 367 (in the RCL), which become more solvent exposedupon RCL displacement, as well as on Tyr185 on the strand s3A, whichis close (below 3 Å) to Glu343 on the strand s5A in the native NS,while such a distance increases to 5 Å in the other twomonomeric con-formers. However, the action of the negatively-charged carboxyl groupof Glu343 as a competitor for the Tyr hydrogen is not the most likely,given the intrinsic low pK of glutamic acid. The tyrosinate band is also

Table 1Secondary structure analysis. Residues are assigned to the main secondary structure ele-ments of the differentNS conformers based on analysis of the crystallographic coordinates,MD simulations and CD data.

α-Helix β-Sheet Other

Native 111 ± 3 108 ± 6 188 ± 7 in vitro 1

107 ± 7 115 ± 7 185 ± 14 in silico 2

100 118 189 ⁎ in crystal 3

116 119 172 ⁎ in crystal 4

Cleaved 087 ± 2 139 ± 3 181 ± 4 in vitro 1

106 ± 7 135 ± 7 166 ± 14 in silico 2

108 145 154 ⁎ in crystal 5

Latent 081 ± 2 138 ± 4 188 ± 5 in vitro 1

102 ± 6 125 ± 6 180 ± 12 in silico 2

Polymer 094 ± 2 118 ± 4 195 ± 6 in vitro 1

⁎ Including not resolved residues.1 Calculated by CDPro from CD spectra.2 Calculated by STRIDE from MD simulation.3 Calculated by STRIDE from PDB ID: 3F5N [15].4 Calculated by STRIDE from PDB ID: 3FGQ [27].5 Calculated by STRIDE from PDB ID: 3F02 [15].

relevant in the polymeric NS, however it is not possible to speculateabout the polymer model in the absence of structural details.

3.4. MD simulations: starting coordinates and latent modeling

All-atomMD simulationswere performed on the three NS states sol-vated with water molecules. The initial molecular structures for MDsimulations were based on the X-ray crystallographic coordinates ofNS by Ricagno et al. [15] for the native (PDB ID: 3F5N) and cleavedNSs (PDB ID: 3F02), and on analogy with the AAT structures [49,50](for details see Appendix A, Supporting Table A1). Since no crystal struc-ture is available for the NS latent form, we modeled the latent NS in acleaved-like conformation, that is according to the cleaved NS crystalstructure [15], with the full insertion of the RCL into the A β-sheet asstrand s4A (Appendix A, Supporting Fig. A5). The dangling, presumablydisordered segment, composed by the s1C strand and part of the RCL,was modeled according to a crystallized latent form of AAT (PDB ID:1IZ2) [30]. A 3D molecular model for the latent NS is reported inAppendix A along with 3D models for native and cleaved NSs used inMD simulations.

3.5. MD simulations: NS structure, stability and salt bridges

The three NS conformers displayed a remarkable conformationalstability throughout the 45 ns long trajectories, which remained closeto the crystallographic structures, with no sign of unfolding, and withRoot Mean Square Displacement (RMSD) values in the order of 2.3 Å.

(Appendix A, Supporting Fig. A6). Also the calculated radii of gyra-tion remained closely comparablewith those calculated from the crystalstructures and did not show specific trends (Appendix A, SupportingTable A3).

The secondary structure fractions calculated fromCDspectra (CDPro)were compared with those assigned from the crystallographic coor-dinates, or from the MD simulations by means of the STRIDE algo-rithm [51] (Fig. 2b, Table 1). Within an overall good agreement, it isworth noting that the simulation results lay in between the results ob-tained from the CD spectra and the crystallographic data, in keepingwith the concept that MD simulations help in relaxing part of the con-straints posed by packing in the crystal lattice. A slight overestimate ofthe α-helix content of cleaved and latent NSs both in the in silico andin crystal analyses is likely due to a correlated underestimate of disor-dered structures, which are not resolved in the crystal structure. Theslightly lower content in the β-sheet structure (about 10 residues) ofthe latent form with respect to the cleaved one, although being at thelimit of uncertainty, may be ascribed to partial unfolding of strand s1Cand to the loss of structure at the ends of strands s5A and s4A.

Time-averaged residue-based Root Mean Square Fluctuations(RMSF), calculated after alignment of the MD generated structureswith the average structure, are color-mapped onto theprotein structures

pdb:3F02pdb:3F02pdb:3F02image of Fig.�4

Fig. 6. Comparison of structural features for the three NS forms, calculated from the MDsimulated models. (a) Solvent Accessible Surface Area (SASA) calculated for all, nonpolarand polar atoms. (b) Number of hydrogen bonds (HB) with the N50% occupancy for total,backbone and side-chain HB.

115R. Noto et al. / Biochimica et Biophysica Acta 1854 (2015) 110–117

in Fig. 5. Apart from turns, termini and loops, which are trivially flexiblein all conformers, native NS shows a slightly higher conformational flex-ibility, as expected in view of the larger number of non-native contacts(Appendix A, Supporting Table A3). Such a flexibility is notably relatedto the D and E helices, in keepingwith the fact that these regions are dis-ordered in the crystal structures [15,28].

A rationale for such differentflexibility can be found in the stable saltbridges, represented as colored spheres in Fig. 5, to highlight theirspatial pattern. Indeed, the cleaved and latent conformers show a con-tinuous chain of salt bridges involving the D helix, which anchor differ-ent regions andmay dampfluctuations (Fig. 5). An additional noticeablecluster of salt bridges stabilizes the A, I andG helices and is present in allthe three NS conformers, being more extended in cleaved NS togetherwith two complex salt bridges, which are known to play key roles inthe protein stability [52] (Appendix A, Supporting Table A4).

A partial opening of the breach region at the top of s3A and s5Astrands occurs around t = 42 ns in the native NS conformer [36],while no similar events were detected in the trajectories of the cleavedand latent NSs. TheNS breach region is pivotal for themechanismof RCLinsertion, and indeed it is a highly conserved structural feature acrossthe serpin family [53]. Such observation prompted us to extend the rel-ative MD simulation up to about 100 ns, to better sample the associatedconformational rearrangements. This event is accompanied by theformation and disruption of hydrogen bonds and salt bridges both inthe breach region and in other regions of the protein (Appendix A,Supporting Fig. A7). Moreover, Essential Mode Analysis [54] confirmsthat such opening is related to the main protein collective mode; inparticular it highlights a suggestive correlation between opening ofthe breach region and large collective movements of the NS loops(Appendix A, Supporting Fig. A8).

3.6. MD simulations: free-energy changes related to neuroserpininactivation

Solvent exposure of the protein surface for the different NS con-formers was measured by calculating the Solvent Accessible SurfaceArea (SASA) and distinguishing the contribution due to polar atoms(hydrophilic contributions) and non-polar atoms (hydrophobic contri-butions). Surprisingly, the differences among the three conformers aresmall, notwithstanding the important associated conformational

Fig. 5.RMSF of theNS conformers. Cα RMSFmapped onto the averageNS structure colored accorin the right panel of Fig. 1): native (left), cleaved (center), latent (right). The structural elementsscale bar on the right. Note the hD helix flexibility, which is higher in the native than in cleaved(orange), glutamate (cyan), and aspartate (pink).

transitions. Indeed, the maximum difference is in the order of 500 Å2,i.e. corresponding to the exposure of a few residues. Cleaved NS hasboth the lowest non-polar SASA and a very high polar contribution. La-tent NS shows the largest total area valuemainly due to the excess SASAof non-polar atoms, which is reduced in cleaved NS. In general, we findthat some kind of compensating effect levels off the differences thatcould be naively expected. For example, the latent conformer com-pensates the decrease in SASA, due to RCL insertion, with a largersurface exposed by residues in the B and C β-sheets. In particular,unfolding of the s1C strand contributes strongly to its non-polar excesssurface area. We may estimate the free-energy differences due toapolar and polar surfaces by taking the native NS structure as a

ding to displacement during 45 ns, for the threeNS conformers (with helix F on the back asare color-coded fromblue (RMSF: 0.5Å, stable) to red (RMSF: 2.0Å,flexible), as in the colorand latent forms. Themost stable salt bridges are represented as colored spheres: arginine

image of Fig.�6

116 R. Noto et al. / Biochimica et Biophysica Acta 1854 (2015) 110–117

reference state, and referring to the empirical expression [55]:ΔGSASA=σapolΔSASAapol − σpolΔSASApol, where σapol = 49.6 cal mol−1 Å−2 andσpol = 19.1 cal mol−1 Å−2. Due to their more favorable SASA, thecleaved and latent NSs are more stable than the native NS, with the fol-lowing free energy contributions ΔGSASA(cleaved) = −14.4 kcal mol−1and ΔGSASA(latent) = −6.5 kcal mol−1.

Hydrogen bonds (HBs) play an important role in proteinstructural stability and functionality [56,57]. Fig. 6 shows the HBs in-volving backbone or side-chain atoms for the three NS conformers.The differences are small but significant, in particular for the backboneHB, where the cleaved and latent forms show an increased number ofHBs, mainly due to the interaction of the strands s5A and s3A with thenew s4A strand. The latter strands form anti-parallel beta strands,which typically host stronger hydrogen bonds than those in parallelbeta strands, as s3A and s5A are in native NS. An opposite trend isobserved for side-chain HBs, but with differences that fall within theerror bars. If one assigns a value of 0.5 kcal mol−1 for the stabilizingfree energy of each HB [58], the free-energy differences due to HBswith respect to native NS are: ΔGHB(cleaved) = −5.5 kcal mol−1 andΔGHB(latent)=−4.0 kcalmol−1, suggesting that the cleaved and latentNSs are partly stabilized by intra-molecular H-bonding

As a further consideration, it is conceivable that some residue side-chains are restrained when NS assumes a less flexible conformation,thus decreasing the configurational entropy. We calculated such an en-tropic contribution following the approach of Pickett et al. [59], whichconsists in estimating the change in relative surface exposure and inweighting these changes with empirical coefficients on a residue-typebasis [58]. The estimated terms for cleaved and latent NSs amounttoΔGS(cleaved)=1.8 kcalmol−1 andΔGS(latent)=1.3 kcalmol−1, re-spectively. Thus, by bringing together the three free energy contributiondescribed above, we estimate a total free energy gain of ΔG(cleaved)=−18.1 kcal mol−1 for the conversion of native NS into the cleaved con-former, andΔG(latent)=−9.2 kcalmol−1, for the native to latent con-former transition. Although these estimated values are based on a seriesof empirical assumptions, they appear to capture the overall stabilityranking of the three NS conformers, as determined by HD exchangeMS, NMR or optical spectroscopy in thermal and chemical denaturationexperiments [13,17,22,60,61].

4. Conclusions

Native NS, as other serpins, is natively folded in a metastable statethat may convert into other conformational isoforms [16], namely thecleaved NS, resulting from protease cleavage duringNS inhibitory activ-ity, and the latent NS, a dysfunctional conformation inactive for inhibi-tion, typically obtained by RCL insertion into the main NS A β-sheetwithout cleavage. The formation of pathological polymers is also intrin-sically related to serpin metastability, although the actual intermo-lecular assembly model of the NS moieties is still debated [17]. Inthis scenario, we used Molecular Dynamics and optical spectroscopiesto characterize in silico and in vitro the different NS conformers and toassess their structural and dynamical properties, as well as their free-energies.

Characteristic differences were observed in the emission spectraof the various NS conformers, related to the interaction patterns oftyrosine residues. Indeed, when only the NS Trp residues are excitedat 295 nm, minor differences, if any, are observed in the emission spec-tra of the different monomeric conformers. On the contrary, by excitingat 275 nm, where tyrosine residues are also excited, emission spectrayield a clear fingerprint of the different conformers, and in particularallow to distinguish latent from native NS. This observation is particu-larly relevant as it offers a simple spectroscopic tool for the selectionof NS conformers, which is currently performed a posteriori by gel elec-trophoresis. In view of its simplicity and generality, the possibility oftransferring such spectroscopic approach to other serpin molecules ap-pears attractive.

In the absence of a crystal structure for latent NS, MD simulationsand computational modeling enabled us to propose a model for thestructure of latent NS, based on its structural homology betweencleaved NS [15] and latent AAT [30]. Themodel was validated by opticalspectroscopy: indeed, the overall good agreement between secondaryand tertiary structure elements obtained from the in vitro and in silicoanalyses, alongwith the conserved secondary structures observed in la-tent and cleaved NSs, can be considered as a validation of the proposedlatent conformation which was modeled on the basis of the cleaved NSstructure.

The NS structures obtained with Molecular Dynamics simula-tions proved very stable, with no significant deviations from the avail-able crystallographic models [15,28], in keeping with the previousMD study of native NS [36]. Cleaved and latent NSs are more stablethan native NS, with a free energy gain of −18.1 kcal mol−1 and−9.2 kcal mol−1, respectively, as expected for secondary structurechanges in proteins or peptides [62]. Our calculations indicate thatsuch stabilization arises from the hydrogen bond network formedupon RCL insertion, and from differences in polar and apolar SolventAccessible Surface Areas and configurational entropy. The Root MeanSquare Fluctuations analysis evidenced, for latent and cleaved NS, anincreased rigidity in a region involving the D helix (Fig. 6), due to theformation of a network of stable salt bridges.

In summary, the present study proposes a simple non-invasivespectroscopic method to diagnostically distinguish between NS confor-mations. The transferability of this tool to other serpins may be con-siderably useful for biophysical and biochemical studies. The MDsimulation further confirms the small structural differences amongmo-nomeric NS conformations and crucially provide a rationale for their dif-ferent dynamics and thermodynamics stability.

Acknowledgements

We thank S. Caccia, R. Russo, E. Miranda, J. Irving, and D. A. Lomas forhelpful discussions and R. Quagliana for assistance in the artworkpreparation. We acknowledge the CINECA for the availability of highperformance computing resources and support. The financial supportof Telethon Foundation — Italy (Grant no. GGP11057) is gratefullyacknowledged. We also acknowledge the financial support from theUniversity of Palermo (FFR program 2012/2013) and from FondazioneCariplo, Milano, Italy (NOBEL project Transcriptomics and ProteomicsApproaches to Diseases of High Socio-medical Impact: a TechnologyIntegrated Network; and also Grant 2013-0967 Molecular and CellularBases of Serpin Conformational Diseases). The funders had no role instudy design, data collection and analysis, decision to publish, or prepa-ration of the manuscript. The authors have declared that no competinginterests exist.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbapap.2014.10.002.

References

[1] G.A. Hastings, T.A. Coleman, C.C. Haudenschild, S. Stefansson, E.P. Smith, R. Barthlow,S. Cherry, M. Sandkvist, D.A. Lawrence, Neuroserpin, a brain-associated inhibitor oftissue plasminogen activator is localized primarily in neurons. Implications for theregulation of motor learning and neuronal survival, J. Biol. Chem. 272 (1997)33062–33067.

[2] G.A. Silverman, P.I. Bird, R.W. Carrell, F.C. Church, P.B. Coughlin, P.G. Gettins, J.A.Irving, D.A. Lomas, C.J. Luke, R.W. Moyer, P.A. Pemberton, E. Remold-O'Donnell,G.S. Salvesen, J. Travis, J.C. Whisstock, The serpins are an expanding superfamily ofstructurally similar but functionally diverse proteins. Evolution, mechanism of inhi-bition, novel functions, and a revised nomenclature, J. Biol. Chem. 276 (2001)33293–33296.

[3] S. Caccia, S. Ricagno, M. Bolognesi, Molecular bases of neuroserpin function and pa-thology, Biomol. Concepts 1 (2010) 117–130.

http://dx.doi.org/10.1016/j.bbapap.2014.10.002http://dx.doi.org/10.1016/j.bbapap.2014.10.002http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0005http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0005http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0005http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0005http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0005http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0300http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0300http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0300http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0300http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0300http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0300http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0015http://refhub.elsevier.com/S1570-9639(14)00262-3/rf0015

117R. Noto et al. / Biochimica et Biophysica Acta 1854 (2015) 110–117

[4] B.D. Roussel, T.M. Newton, E. Malzer, N. Simecek, I. Haq, S.E. Thomas, M.L. Burr, P.J.Lehner, D.C. Crowther, S.J. Marciniak, D.A. Lomas, Sterol metabolism regulatesneuroserpin polymer degradation in the absence of the unfolded protein responsein the dementia FENIB, Hum. Mol. Genet. 22 (2013) 4616–4626.

[5] K.J. Kinghorn, D.C. Crowther, L.K. Sharp, C. Nerelius, R.L. Davis, H.T. Chang, C. Green,D.C. Gubb, J. Johansson, D.A. Lomas, Neuroserpin binds Aβ and is a neuroprotectivecomponent of amyloid plaques in Alzheimer disease, J. Biol. Chem. 281 (2006)29268–29277.

[6] R.L. Davis, A.E. Shrimpton, P.D. Holohan, C. Bradshaw, D. Feiglin, G.H. Collins, P.Sonderegger, J. Kinter, L.M. Becker, F. Lacbawan, D. Krasnewich, M. Muenke, D.A.Lawrence, M.S. Yerby, C.M. Shaw, B. Gooptu, P.R. Elliott, J.T. Finch, R.W. Carrell,D.A. Lomas, Familial dementia caused by polymerization ofmutant neuroserpin, Na-ture 401 (1999) 376–379.

[7] D. Belorgey, D.C. Crowther, R. Mahadeva, D.A. Lomas, Mutant neuroserpin(Ser49Pro) that causes the familial dementia FENIB is a poor proteinase inhibitorand readily forms polymers in vitro, J. Biol. Chem. 277 (2002) 17367–17373.

[8] D. Belorgey, L.K. Sharp, D.C. Crowther, M. Onda, J. Johansson, D.A. Lomas,Neuroserpin Portland (Ser52Arg) is trapped as an inactive intermediate that rapidlyforms polymers: implications for the epilepsy seen in the dementia FENIB, Eur. J.Biochem. 71 (2004) 3360–3367.

[9] M. Onda, D. Belorgey, L.K. Sharp, D.A. Lomas, Latent S49P neuroserpin forms poly-mers in the dementia familial encephalopathy with neuroserpin inclusion bodies,J. Biol. Chem. 280 (2005) 13735–13741.

[10] E. Miranda, K. Romisch, D.A. Lomas, Mutants of neuroserpin that cause dementia ac-cumulate as polymers within the endoplasmic reticulum, J. Biol. Chem. 279 (2004)28283–28291.

[11] E. Miranda, I. MacLeod, M.J. Davies, J. Perez, K. Romisch, D.C. Crowther, D.A. Lomas,The intracellular accumulation of polymeric neuroserpin explains the severity of thedementia FENIB, Hum. Mol. Genet. 17 (2008) 1527–1539.

[12] R.W. Carrell, D.A. Lomas, Conformational disease, Lancet 350 (1997) 134–138.[13] P.G. Gettins, Serpin structure, mechanism, and function, Chem. Rev. 102 (2002)

4751–4804.[14] J.A. Huntington, R.J. Read, R.W. Carrell, Structure of a serpin–protease complex

shows inhibition by deformation, Nature 407 (2007) 923–926.[15] S. Ricagno, S. Caccia, G. Sorrentino, G. Antonini, M. Bolognesi, Human neuroserpin:

structure and time-dependent inhibition, J. Mol. Biol. 388 (2009) 109–121.[16] J.C. Whisstock, S.P. Bottomley, Molecular gymnastics: serpin structure, folding and

misfolding, Curr. Opin. Struct. Biol. 16 (2006) 761–768.[17] U.I. Ekeowa, J. Freeke, E. Miranda, B. Gooptu, M.F. Bush, J. Perez, J. Teckman, C.V.

Robinson, D.A. Lomas, Defining the mechanism of polymerization in theserpinopathies, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 17146–17151.

[18] D.A. Lomas, D.L. Evans, J.T. Finch, R.W. Carrell, The mechanism of Z alpha 1-antitrypsin accumulation in the liver, Nature 357 (1992) 605–607.

[19] P. Sivasothy, T.R. Dafforn, P.G.W. Gettins, D.A. Lomas, Pathogenic α1-antitrypsinpolymers are formed by reactive loop-β–sheet A linkage, J. Biol. Chem. 275 (2000)33663–33668.

[20] M. Yamasaki, W. Li, D.J. Johnson, J.A. Huntington, Crystal structure of a stable dimerreveals themolecular basis of serpin polymerization, Nature 455 (2008) 1255–1258.

[21] M. Yamasaki, T.J. Sendall, M.C. Pearce, J.C. Whisstock, J.A. Huntington, Molecularbasis of alpha1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer, EMBO Rep. 12 (2011) 1011–1017.

[22] S. Ricagno, M. Pezzullo, A. Barbiroli, M. Manno, M. Levantino, M.G. Santangelo, F.Bonomi, M. Bolognesi, Two latent and two hyperstable polymeric forms of humanneuroserpin, Biophys. J. 99 (2010) 3402–3411.

[23] M.G. Santangelo, R. Noto, M. Levantino, A. Cupane, S. Ricagno, M. Pezzullo, M.Bolognesi, M.R. Mangione, V. Martorana, M. Manno, On the molecular structure ofhuman neuroserpin polymers, Proteins 80 (2012) 8–13.

[24] D. Belorgey, P. Hagglof, M. Onda, D.A. Lomas, pH dependent stability of neuroserpinis mediated by histidines 119 and 138; implications for the control of β-sheet A andpolymerization, Methods 53 (2010) 255–266.

[25] R. Noto, M.G. Santangelo, S. Ricagno, M.R. Mangione, M. Levantino, M. Pezzullo, V.Martorana, A. Cupane, M. Bolognesi, M. Manno, The tempered polymerization ofhuman neuroserpin, PLoS One 7 (2012) e32444.

[26] A. Chiou, P. Hagglof, A. Orte, A.Y. Chen, P.D. Dunne, D. Belorgey, S. Karlsson-Li, D.A.Lomas, D. Klenerman, Probing neuroserpin polymerization and interaction withamyloid-β peptides using single molecule fluorescence, Biophys. J. 97 (2009)2306–2315.

[27] T.R. Dafforn, R. Mahadeva, P.R. Elliott, P. Sivasothy, D.A. Lomas, A kinetic mechanismfor the polymerization of α1-antitrypsin, J. Biol. Chem. 274 (1999) 9548–9555.

[28] S. Takehara, M. Onda, J. Zhang, M. Nishiyama, X. Yang, B. Mikami, D.A. Lomas, The 2.1-Å crystal structure of native neuroserpin reveals unique structural elements thatcontribute to conformational instability, J. Mol. Biol. 388 (2009) 11–20.

[29] B. Gooptu, D.A. Lomas, Conformational pathology of the serpins: themes, variationsand therapeutic strategies, Annu. Rev. Biochem. 78 (2009) 147–176.

[30] H. Im, M.S. Woo, K.Y. Hwang, M.H. Yu, Interactions causing the kinetic trap in serpinprotein folding, J. Biol. Chem. 277 (2002) 46347–46354.

[31] Q. Zhang, A.M. Buckle, R.H. Law, M.C. Pearce, L.D. Cabrita, G.J. Lloyd, J.A. Irving, A.I.Smith, K. Ruzyla, J. Rossjohn, S.P. Bottomley, J.C. Whisstock, The N terminus of theserpin, tengpin, functions to trap the metastable native state, EMBO Rep. 8 (2007)658–663.

[32] L. Beinrohr, V. Harmat, J. Dobo, Z. Lorincz, P. Gal, P. Zavodszky, C1 inhibitor serpindomain structure reveals the likely mechanism of heparin potentiation and confor-mational disease, J. Biol. Chem. 282 (2007) 21100–21109.

[33] T.J. Stout, H. Graham, D.I. Buckley, D.J. Matthews, Structures of active and latent PAI-1: a possible stabilizing role for chloride ions, Biochemistry 39 (2000) 8460–8469.

[34] B. Gooptu, B. Hazes, W.S.W. Chang, T.R. Dafforn, R.W. Carrell, R.J. Read, D.A. Lomas,Inactive conformation of the serpin α1-antichymotrypsin indicates two-stage inser-tion of the reactive loop: implications for inhibitory function and conformationaldisease, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 67–72.

[35] S. Takehara, J. Zhang, X. Yang, N. Takahashi, B. Mikami, M. Onda, Refolding and po-lymerization pathways of neuroserpin, J. Mol. Biol. 403 (2010) 751–762.

[36] A. Sarkar, C. Zhou, R. Meklemburg, P. Wintrode, Local conformational flexibility pro-vides a basis for facile polymer formation in human neuroserpin, Biophys. J. 101(2011) 1758–1765.

[37] D. Belorgey, J.A. Irving, U.I. Ekeowa, J. Freeke, B.D. Rouseel, E. Miranda, J. Pérez, C.V.Robinson, S.J. Marciniak, D.C. Crowther, C.H. Michel, D.A. Lomas, Characterizationof serpin polymers in vitro and in vivo, Methods 53 (2011) 255–266.

[38] S.M. Kelly, T.J. Jess, N.C. Price, How to study proteins by circular dichroism, Biophys.Biochem. Acta 1751 (2005) 119–139.

[39] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic/PlenumPublishers, New York, 1999.

[40] J.A. Gardecki, M. Maroncelli, Set of secondary emission standards for calibration of thespectral responsivity in emission spectroscopy, Appl. Spectrosc. 52 (1998) 1179–1189.

[41] A.D.MacKerell Jr., D. Bashford, M. Bellott, R.L. Dunbrack Jr., J.D. Evanseck, M.J. Field, S.Fischer, J. Gao, H. Guo, S. Ha, D. Joseph-McCarthy, L. Kuchnir, K. Kuczera, F.T.K. Lau, C.Mattos, S. Michnick, T. Ngo, D.T. Nguyen, B. Prodhom, W.E. Reiher III, B. Roux, M.Schlenkrich, J.C. Smith, R. Stote, J. Straub, M. Watanabe, J. Wiorkiewicz-Kuczera, D.Yin, M. Karplus, All-atom empirical potential for molecular modeling and dynamicsstudies of proteins, J. Phys. Chem. B 102 (1998) 3586–3616.

[42] J.C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot, R.D.Skeel, L. Kalé, K. Schulten, Scalable molecular dynamics with NAMD, J. Comput.Chem. 26 (2005) 1781–1802.

[43] N. Sreerama, R.W. Woody, Estimation of protein secondary structure from circulardichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with anexpanded reference set, Anal. Biochem. 287 (2000) 252–260.

[44] Y.K. Reshetnyak, E.A. Burstein, Decomposition of protein tryptophan fluorescencespectra into log-normal components. II. The statistical proof of discreteness of tryp-tophan classes in proteins, Biophys. J. 81 (2001) 1710–1734.

[45] J.L. Meagher, J.M. Beechem, S.T. Olson, P.G.W. Gettins, Deconvolution of the fluores-cence emission spectrum of human antithrombin and identification of the trypto-phan residues that are responsive to heparin binding, J. Biol. Chem. 273 (1998)23283–23289.

[46] D.J. Tew, S.P. Bottomley, Probing the equilibrium denaturation of the serpin a1-antitrypsin with single tryptophan mutants; evidence for structure in the urea un-folded state, J. Mol. Biol. 313 (2001) 1161–1169.

[47] M.C. Pearce, L.D. Cabrita, A.M. Ellisdon, S.P. Bottomley, The loss of tryptophan 194 inantichymotrypsin lowers the kinetic barrier to misfolding, FEBS J. 274 (2007)3622–3632.

[48] J. Eisinger, B. Feuer, A.A. Lamola, Intramolecular singlet excitation transfer. Applica-tions to polypeptides, Biochemistry 8 (1969) 3908–3915.

[49] P.R. Elliott, X.Y. Pei, T.R. Dafforn, D.A. Lomas, Topography of a 2.0 Å structure of α1-antitrypsin reveals targets for rational drug design to prevent conformational dis-ease, Protein Sci. 9 (2000) 1274–1281.

[50] M.A. Dunstone, W. Dai, J.C. Whisstock, J. Rossjohn, R.N. Pike, et al., Cleavedantitrypsin polymers at atomic resolution, Protein Sci. 9 (2000) 417–420.

[51] D. Frishman, P. Argos, Knowledge-based protein secondary structure assignment,Proteins Struct. Funct. Genet. 23 (1995) 566–579.

[52] A.G. Gvritishvili, A.V. Gribenko, G.I. Makhatadze, Cooperativity of complex salt brid-ges, Protein Sci. 17 (2008) 1285–1290.

[53] J.A. Irving, R.N. Pike, A.M. Lesk, J.C. Whisstock, Phylogen of the serpin superfamily:implications of patterns of amino acid conservation for structure and function,Genom. Res. 10 (2000) 1845–1864.

[54] H.J. Berendsen, S. Hayward, Collective protein dynamics in relation to function, Curr.Opin. Struct. Biol. 10 (2000) 165–169.

[55] Z.P. Weng, C. Delisi, S. Vajda, Empirical free energy calculation: comparison to calo-rimetric data, Protein Sci. 6 (1997) 1976–1984.

[56] G.D. Rose, R. Wolfenden, Hydrogen bonding, hydrophobicity, packing, and proteinfolding, Annu. Rev. Biophys. Biomol. Struct. 22 (1993) 381–415.

[57] D. Shortle, Denatured states of proteins and their roles in folding and stability, Curr.Opin. Struct. Biol. 3 (1993) 66–74.

[58] S. Kumar, R. Nussinov, Close-range electrostatics interactions in proteins,Chembiochem 3 (2002) 604–617.

[59] S.D. Pickett, M.J. Sternberg, Empirical scale of side-chain conformational entropy inprotein folding, J. Mol. Biol. 231 (1993) 825–839.

[60] T.R. Dafforn, R.N. Pike, S.P. Bottomley, Physical characterization of serpin con-formers, Methods 32 (2004) 150–158.

[61] Y. Tsutsui, A. Sarkar, P.L. Wintrode, Probing serpin conformational change usingmass spectrometry and related methods, Methods Enzymol. 501 (2011) 325–350.

[62] R. Carrotta, M. Manno, D. Bulone, V. Martorana, P.L. San Biagio, Protofibril formationof amyloid b-protein at low pH via a non-cooperative elongation mechanism, J. Biol.Chem. 280 (2005) 30001–30008.

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Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics1. Introduction2. Materials and methods2.1. Production of different neuroserpin conformers2.2. Circular dichroism2.3. Intrinsic fluorescence2.4. Molecular Dynamics simulations2.5. Computational analytical tool

3. Results and discussion3.1. Characterization of NS secondary structure and validation of the latent model3.2. Characterization of NS overall structure and identification of the latent conformation3.3. Tyrosine emission spectra and interaction network3.4. MD simulations: starting coordinates and latent modeling3.5. MD simulations: NS structure, stability and salt bridges3.6. MD simulations: free-energy changes related to neuroserpin inactivation

4. ConclusionsAcknowledgementsAppendix A. Supplementary dataReferences